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Abstract

Although hyperaldosteronemia exerts detrimental impacts on vascular endothelium in addition to elevating blood pressure, the effects and molecular mechanisms of hyperaldosteronemia on early endothelial progenitor cell (EPC)–mediated endothelial repair after arterial damage are yet to be determined. The aim of this study was to investigate the endothelial repair capacity of early EPCs from hypertensive patients with primary hyperaldosteronemia (PHA). In vivo endothelial repair capacity of early EPCs from PHAs (n=20), age- and blood pressure–matched essential hypertension patients (n=20), and age-matched healthy subjects (n=20) was evaluated by transplantation into a nude mouse carotid endothelial denudation model. Endothelial function was evaluated by flow-mediated dilation of brachial artery in human subjects. In vivo endothelial repair capacity of early EPCs and flow-mediated dilation were impaired both in PHAs and in essential hypertension patients when compared with age-matched healthy subjects; however, the early EPC in vivo endothelial repair capacity and flow-mediated dilation of PHAs were impaired more severely than essential hypertension patients. Oral spironolactone improved early EPC in vivo endothelial repair capacity and flow-mediated dilation of PHAs. Increased oxidative stress, oxidative 5,6,7,8-tetrahydrobiopterin degradation, endothelial nitric oxide synthase uncoupling and decreased nitric oxide production were found in early EPCs from PHAs. Nicotinamide adenine dinucleotide phosphate oxidase subunit p47phox knockdown or 5,6,7,8-tetrahydrobiopterin supplementation attenuated endothelial nitric oxide synthase uncoupling and enhanced in vivo endothelial repair capacity of early EPCs from PHAs. In conclusion, PHAs exhibited more impaired endothelial repair capacity of early EPCs than did essential hypertension patients independent of blood pressure, which was associated with mineralocorticoid receptor–dependent oxidative stress and subsequently 5,6,7,8-tetrahydrobiopterin degradation and endothelial nitric oxide synthase uncoupling.

Introduction

Vascular endothelial injury contributes to the initiation and progression of atherosclerotic vascular disease.1–3 It has been demonstrated that early endothelial progenitor cells (EPCs) play a pivotal role in endothelial repair process.4–10 However, functional impairment of early EPCs observed in patients with several cardiovascular risk factors leads to decreased endothelium-reparative capacity and increased incidence of atherosclerotic vascular disease.6,7,9–11 Therefore, understanding novel mechanisms of early EPC dysfunction related to cardiovascular risk factors has important clinical relevance for the prevention and treatment of atherosclerotic vascular disease.

Primary hyperaldosteronemia patients (PHAs) displayed an increased rate of cardiovascular events when compared with essential hypertension patients (EHs) with similar blood pressure (BP) level,12 suggesting potential pathological effects of hyperaldosteronemia on cardiovascular system in addition to elevation of BP. Indeed, accumulating evidence indicates that hyperaldosteronemia has harmful effects on blood vessels via the direct action of aldosterone independent of rising BP.13,14 Recently, it has been reported that high level of aldosterone inhibited the early EPC formation from bone marrow precursor cells15 and vascularization capacity,16 suggesting that hyperaldosteronemia might also influence the early EPC-mediated endogenous repair mechanism apart from the direct detrimental effect on vascular wall. However, the in vivo effect of hyperaldosteronemia on early EPC function, especially the endothelial repair capacity, needed to be further determined. Therefore, in the present study, we compared the early EPC in vivo endothelial repair capacity of PHAs to age- and BP-matched EHs and age-matched healthy subjects (HSs) and analyzed the underlying molecular mechanism.

It has been demonstrated that aldosterone is a strong mediator stimulating oxidative stress in vascular cells.17 Oxidative stress–led 5,6,7,8-tetrahydrobiopterin (BH4) degradation is the main pathogenic cause of endothelial nitric oxide synthase (eNOS) uncoupling.18,19 The eNOS uncoupling has been proved as an important molecular mechanism underlying atherosclerotic vascular disease.19,20 On the basis of these data, we hypothesized that oxidative BH4 degradation and eNOS uncoupling may involve in the hyperaldosteronemia-caused alteration of early EPC function. Therefore, in the present study, we attempted to clarify the role of oxidative stress and BH4 bioavailability and eNOS/NO pathway in the regulation of early EPC-mediated endothelial repair in PHAs.

Methods

Study Subjects

Twenty patients identified with an aldosterone-producing adenoma, thus PHAs based on increased aldosterone/renin ratio (>50) and pathological saline infusion test and imaging test via high-resolution computed tomographic scan were recruited. All the patients were retrospectively defined to aldosterone-producing adenoma by histopathology after surgical treatment. All the antihypertensive drugs were stopped >2 weeks for the diagnostic tests before recruitment. After recruitment, all the PHAs were treated with spironolactone (60–120 mg/d) for 4 to 6 weeks before surgical treatment. Twenty newly diagnosed EHs without pharmacological therapy randomly matched for age and BP to enlisted PHAs were recruited from patients visited in the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). Twenty age-matched HSs were recruited as the controls. The PHAs and EHs were included without other known cardiovascular disease, and the included HSs had no cardiovascular risk factors. All the study subjects were excluded diabetes mellitus, malignant disease, infection, or inflammatory disorders. BP measurements were performed according to the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. All of the subjects underwent 3 BP measurements in 2 different visits, after 30 minutes of rest, and the measurements were spaced by 5- to 10-minute intervals, on both the left and right arm, in the sitting and lying positions. The clinical characteristics are summarized in Table S1 in the online-only Data Supplement. Our study was confirmed to the ethical principles outlined in the Declaration of Helsinki. The experimental protocol was approved by the Ethical Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China), and written informed consent was obtained from every study participant.

Statistical Analysis

All results are expressed as mean±SEM. Comparisons between groups were analyzed by the Student t test. Statistical analysis was performed by a 1-way ANOVA, followed by multiple comparisons using either the Student–Newman–Keuls or Tukey–Kramer test. A value of P<0.05 was considered to denote statistical significance. All statistical analyses used SPSS statistical software (SPSS version 13.0).

Other detailed methods performed in the present study are provided in the online-only Data Supplement.

Results

In Vivo Endothelial Repair Capacity of Early EPCs From PHAs, EHs, and HSs

In vivo endothelial repair area was evaluated by Evans Blue staining, and the representative photographs are shown in Figure 1B. Transplantation of early EPCs accelerated in vivo endothelial repair of denudated injured carotid artery markedly compared with PBS injection (Figure 1A). In vivo endothelial repair capacity of early EPCs from PHAs and EHs was obviously impaired when compared with HSs (Figure 1A). The in vivo endothelial repair capacity of early EPCs from PHAs was impaired more severely than from EHs (Figure 1A). After oral spironolactone for 4 to 6 weeks, in vivo endothelial repair capacity of early EPCs from PHAs was significantly improved (Figure 1A). Phycoerythrin-conjugated monoclonal mouse anti-human CD14 antibody-labeled early EPCs were detected homing to the sites of the injured carotid arteries but not in the contralateral uninjured carotid arteries via both the confocal laser scanning microscopy analysis (Figure S2A) and the fluorescence-activated cell sorter analysis (Figure S2B). The cultured human umbilical vein endothelial cells were injected into the tail vein of nude mice with carotid injury as the negative control. After human umbilical vein endothelial cells injection, little phycoerythrin-conjugated monoclonal mouse anti-human CD14 antibody-labeled cells were detected homing to the sites of the injured carotid arteries via the confocal laser scanning microscopy analysis and fluorescence-activated cell sorter analysis (data not shown). Moreover, human umbilical vein endothelial cells injection did not improve endothelial repair (Figure S2C).

In vivo endothelial repair capacity of early endothelial progenitor cells (EPCs) and relation to endothelial function. A, Endothelial repair at day 3 after carotid injury in nude mice with PBS injection (n=20) or transplantation of early EPCs from healthy subjects (HSs; n=20), essential hypertension patients (EHs; n=20), and primary hyperaldosteronemia patients (PHAs; n=20) before or after treatment with spironolactone (Spi). B, Representative photographs: denudated-endothelium area was stained in blue; endothelial repair area in white. C, Endothelial function was evaluated by flow-mediated dilation (FMD) expressed as the percentage change of the brachial artery diameter. D, Relationship between early EPC in vivo endothelial repair capacity and FMD of the subjects (, HSs, n=20; ◆, EHs, n=20; ▲, PHAs, n=20; ●, PHAs after treatment with Spi, n=20).

In vitro early EPC function, such as migration to vascular endothelial growth factor/stromal cell–derived factor-1 and adhesion to fibronectin in flow, was also evaluated in the present study. As the results shown in Figure S4, in vitro migration and adhesion activity of early EPCs were impaired obviously in PHAs and EHs when compared with HSs, and a further impairment was shown in PHAs compared with EHs. The in vitro migration and adhesion activity of early EPCs in PHAs were obviously promoted after oral spironolactone treatment (Figure S4). The number of circulating EPCs (as assessed by CD34/kinase-insert domain receptor double-positive or CD133/kinase-insert domain receptor double-positive peripheral blood mononuclear cells via fluorescence-activated cell sorter) has no significant difference from PHAs, EHs, and HSs (Figure S5); similar to previous study,16 data reported here suggested that hyperaldosteronemia leads to qualitative rather than quantitative impairment on circulating EPCs.

Endothelial Function in PHAs, EHs, and HSs

Endothelial function expressed as the flow-mediated dilation (FMD) of brachial artery was obviously reduced in PHAs and EHs when compared with HSs (Figure 1C), and FMD was reduced more in PHAs than in EHs (Figure 1C). After oral spironolactone for 4 to 6 weeks, the FMD in PHAs was markedly improved (Figure 1C). In vivo endothelial repair capacity of early EPCs was positively related to FMD (Figure 1D).

Increased Oxidative Stress in Early EPCs From PHAs

Oxidative stress level of early EPCs was evaluated by intracellular reactive oxygen species (ROS) production via 2′,7′-dichlorodihydrofluorescein diacetate fluorescence and superoxide production via electron paramagnetic resonance spectroscopy analysis, respectively. Intracellular ROS production (Figure 2A) and superoxide production (Figure 2B and 2C) were obviously increased in early EPCs from PHAs when compared with HSs. After oral spironolactone for 4 to 6 weeks, both the intracellular ROS production (Figure 2A) and the superoxide production (Figure 2B and 2C) were reduced in early EPCs from PHAs.

Reactive oxygen species (ROS) and superoxide production, p47phox transcription and membrane localization. A, Semiquantitative analysis of intracellular ROS production via 2′,7′-dichlorodihydrofluorescein diacetate (DCF) fluorescence intensity in early endothelial progenitor cells (EPCs) from healthy subjects (HSs; n=12) and primary hyperaldosteronemia patients (PHAs; n=10) before or after treatment with spironolactone (Spi). B, Electron paramagnetic resonance (EPR) spectroscopy analyses of superoxide production of early EPCs from HSs (n=8) and PHAs (n=10) before or after treatment with Spi. C, The representative EPR spectra of superoxide production. D, The p47phox transcription of early EPCs from HSs (n=12) and PHAs (n=10) before or after treatment with Spi was detected by real-time reverse transcriptase-polymerase chain reaction. The GAPDH was used as the internal control. E, The membrane and cytosol fractions of p47phox protein were analyzed by immunoblotting in early EPCs from HSs (n=12) and PHAs (n=10) before or after treatment with Spi. Total cell lysate immunoblotted with actin for control.

Increased p47phox Transcription and Translocation to Plasma Membrane in Early EPCs From PHAs

The important nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p47phox transcription of early EPCs from PHAs was obviously increased compared with HSs (Figure 2D). The membrane localization of p47phox protein was also increased in early EPCs from PHAs when compared with HSs (Figure 2E). After oral spironolactone for 4 to 6 weeks, p47phox transcription and translocation of early EPCs from PHAs were obviously inhibited (Figure 2D and 2E). The transcription and expression of NADPH oxidase isoforms (NOX1, NOX2, and NOX4) in early EPCs showed no apparent difference between the HSs and the PHAs (Figure S6); the result was consistent with the in vitro effect of aldosterone on endothelial cells reported by Nagata et al21 previously.

Decreased NO Generation in Early EPCs From PHAs

NO generation was evaluated both as intracellular cGMP concentration via ELISA system and l-arginine/l-citrulline conversion via high-performance liquid chromatography system analysis. Our data showed that NO generation was obviously decreased in early EPCs from PHAs when compared with HSs (Figure 3A and 3B). After oral spironolactone for 4 to 6 weeks, NO generation was restored in early EPCs from PHAs (Figure 3A and 3B). The representative chromatograms of high-performance liquid chromatography for l-arginine/l-citrulline conversion are shown in Figure 3C.

Nitric oxide (NO) production. A, NO production evaluated as intracellular cGMP concentration via an ELISA system in early endothelial progenitor cells (EPCs) from healthy subjects (HSs; n=12) and primary hyperaldosteronemia patients (PHAs; n=10) before or after treatment with spironolactone (Spi). B, Intracellular l-arginine/l-citrulline conversion was assayed by high-performance liquid chromatography system to express NO generation in early EPCs from HSs (n=8) and PHAs (n=10) before or after treatment with Spi. C, (a) Chromatogram of the mixture of standard l-citrulline and l-arginine. (b) Chromatogram of the extract of early EPCs from HSs. (c) Chromatogram of the extract of early EPCs from PHAs. (d) Chromatogram of the extract of early EPCs from PHAs after treatment with Spi. The individual peaks marked are (1) citrulline and (2) arginine.

Oxidative BH4 Degradation and eNOS Uncoupling in Early EPCs From PHAs

BH4 plays a crucial role as a cofactor of eNOS for generating NO. In its absence, eNOS produces superoxide rather than NO,19 which was reported to mechanistically correlate with EPC dysfunction.22 Therefore, we detected the intracellular biopterin content via high-performance liquid chromatography system and analyzed the amount of BH4 in early EPCs. There was no obvious difference of the total biopterin content in early EPCs from PHAs and HSs (Figure 4A and 4B). In the early EPCs from PHAs compared with HSs, the BH4 content was substantially reduced, and the oxidized forms of BH4 (BH2 and biopterin) were correspondingly increased (Figure 4A and 4B). After oral spironolactone for 4 to 6 weeks, oxidation of BH4 in early EPCs from PHAs was obviously attenuated and BH4 content was significantly increased (Figure 4A and 4B).

5,6,7,8-tetrahydrobiopterin (BH4) level and endothelial nitric oxide synthase (eNOS) dimerization. A, The representative chromatograms of high-performance liquid chromatography (HPLC) for intracellular biopterin measurement in early endothelial progenitor cells (EPCs). B, Intracellular biopterin content was detected by HPLC system in early EPCs from healthy subjects (HSs; n=12) and primary hyperaldosteronemia patients (PHAs; n=10) before or after treatment with spironolactone (Spi). *P<0.01 vs BH4 content of early EPCs from HSs, **P<0.01 vs oxidized biopterin content of early EPCs from HSs, #P<0.01 vs BH4 content of early EPCs from PHAs before Spi treatment, ##P<0.01 vs oxidized biopterin content of early EPCs from PHAs before Spi treatment. C, The eNOS dimerization of early EPCs from HSs (n=12) and PHAs (n=10) before or after treatment with Spi was analyzed by low-temperature SDS-PAGE and immunoblotting; quantification expressed as eNOS dimer/monomer rate.

The eNOS uncoupling-dependent impairment of NO production was mechanistically involved in EPC dysfunction,22 and the decreased eNOS dimer/monomer ratio can be served as a marker of eNOS uncoupling.21,22 Therefore, we performed low-temperature SDS-PAGE and immunoblotting to investigate eNOS dimerization. There was no difference in total eNOS expression in early EPCs from PHAs and HSs using conventional SDS-PAGE (Figure 4C), but low-temperature SDS-PAGE showed that the eNOS dimer/monomer ratio was decreased in early EPCs from PHAs compared with HSs (Figure 4C). After oral spironolactone for 4 to 6 weeks, the eNOS dimer/monomer ratio in early EPCs from PHAs was markedly increased (Figure 4C).

P47phox-siRNA Improved In Vivo Endothelial Repair Capacity of Early EPCs From PHAs

Treatment with p47phox-siRNA decreased the transcription and expression of p47phox in early EPCs from PHAs obviously (Figure S7). Knockdown of p47phox via siRNA attenuated oxidative stress level (Figure 5A and 5B) and BH4 oxidation (Figure 5E) and eNOS uncoupling (Figure 5F), as well as increased NO generation (Figure 5C and 5D) of early EPCs from PHAs. Importantly, p47phox-siRNA significantly improved in vivo endothelial repair capacity of early EPCs from PHAs (Figure 5G and 5H). Correspondingly, the in vitro migration and adhesion activity of early EPCs from PHAs were promoted by p47phox-siRNA (Figure S8).

BH4 Promoted In Vivo Endothelial Repair Capacity of Early EPCs From PHAs

Although BH4 treatment (1 mmol/L) did not change ROS production (data not shown) and oxidative BH4 degradation (Figure 6A) of early EPCs from PHAs, it increased the total biopterin and BH4 content (Figure 6A) of early EPCs from PHAs. Correspondingly, BH4 treatment was able to attenuated eNOS uncoupling (Figure 6B) and increased NO generation (Figure 6C and 6D), as well as promoted in vivo endothelial repair capacity (Figure 6E and 6F) of early EPCs from PHAs. Moreover, the in vitro migration and adhesion activity of early EPCs from PHAs were also enhanced by BH4 treatment (Figure S8).

Discussion

The present study demonstrated first that in vivo endothelial repair capacity of early EPCs was severely impaired in PHAs accompanied with endothelial dysfunction. Increased oxidative stress, oxidative BH4 degradation, eNOS uncoupling, and reduced NO generation were observed to mechanistically correlate with impaired in vivo endothelial repair capacity of early EPCs from PHAs. Oral mineralocorticoid receptor (MR) antagonist spironolactone improved the early EPC in vivo endothelial repair capacity and endothelial function of PHAs. Knockdown of the NADPH oxidase subunit p47phox via siNRA or BH4 treatment was able to restore early EPC in vivo endothelial repair capacity of PHAs.

Abnormalities in endothelial structure and function thought as important processes underlying the development of atherosclerotic vascular disease are likely the result of imbalance between endothelial damage and repair.23,24 The early EPCs, also termed as monocytic EPCs or circulating angiogenic cells,25 have been proved homing to injured vascular wall and promoting endothelial repair by direct and indirect mechanism in our8–10 and other previous studies.6,7 To investigate the homing and endothelial repair capacity of early EPCs derived from clinical subjects in vivo, the athymic nude mice with carotid endothelial denuded injury were introduced to accept human early EPC transplantation in the present study. This nude mouse model was usually used in experiments for EPC transplantation research; the data of our and other previous studies6–10 indicated that this nude mouse model can nearly avoid species rejection and satisfy the EPC transplantation research. In the present study, the confocal laser scanning microscopy analysis showed a subendothelial homing of intravenously injected early EPCs on the endothelial repair zone of the injured carotid artery, what the result in line with the recent study by Giannotti et al7 suggested that early EPCs promote the endothelial repair process likely, in particular, by paracrine mechanisms. However, the beneficial effect of early EPCs for endothelial repair was limited when the functional impairment occurred in the presence of some cardiovascular risk factors.11 Raising BP regardless of whether the cause is essential or secondary to endocrine or renal processes is positively correlated with the incidence of cardiovascular events and leads to endothelial injury. Impaired endothelial repair capacity of early EPCs accompanied by endothelial dysfunction in EHs has been reported previously.7 The present study provided the first evidence that in vivo endothelial repair capacity of early EPCs was also impaired in secondary hypertensive patients with PHA, suggesting a novel pathological mechanism for vascular complications caused by hyperaldosteronemia. To further investigate the possible difference between the EHs and the PHAs on the early EPC-based endogenous endothelial repair capacity, we compared the in vivo endothelial repair capacity of early EPCs from PHAs to the age- and BP-matched EHs in the present study. We found that in vivo endothelial repair capacity of early EPCs from EHs was impaired when compared with HSs similar with previous study,7 but the early EPCs from PHAs showed a more severely impaired in vivo endothelial repair capacity than from EHs. Correspondingly, a further reduced FMD was showed in PHAs when compared with age- and BP-matched EHs. These results provided further evidence to prove that impaired early EPC-mediated endogenous endothelial repair capacity contributes to endothelial dysfunction and proposed a novel mechanism to explain the increased rate of cardiovascular events in PHAs when compared with EHs independent of BP.

Increasing evidence indicates that acute and chronic oxidative stress in cardiovascular system is an essential molecular mechanism underlying the development of atherosclerotic vascular disease.26 Previous studies reported that aldosterone treatment in vitro induced ROS production of early EPCs.15,16 In the present study, we observed that the intracellular ROS and superoxide production were obviously increased in early EPCs from PHAs, which further proved that the high level of aldosterone induced oxidative stress of early EPCs in vivo. In endothelial cells, aldosterone-induced oxidative stress increases oxidative degradation of BH4 that leads to eNOS uncoupling and subsequently decreased NO generation.21 The eNOS uncoupling has also been reported to contribute to EPC functional impairment.22 The oxidative BH4 degradation and eNOS uncoupling and decreased NO generation were showed in early EPCs from PHAs in the present study as well, suggesting that oxidative BH4 degradation and derangement of eNOS/NO pathway may be also involved in the functional impairment of early EPCs in PHAs.

One main source of superoxide in vasculature stimulated by aldosterone is the NADPH oxidase.27 Nagata et al21 have recently proved that p47phox subunit of the NADPH oxidase plays an important role in aldosterone-stimulated ROS production in endothelial cells.21 The present study showed an obviously increased p47phox transcription and translocation to the plasma membrane fraction in early EPCs from PHAs, indicating the NADPH oxidase activation. To further investigate the connection between the increased ROS production and the NADPH oxidase activation, we treated early EPCs of PHAs with p47phox-siRNA or NADPH oxidase inhibitor apocynin. It showed clearly that either p47phox-siRNA or apocynin markedly inhibited the oxidative stress of early EPCs from PHAs. In parallel, BH4 oxidation and eNOS uncoupling were attenuated, and NO generation was increased in early EPCs from PHAs by p47phox-siRNA. Importantly, p47phox-siRNA restored in vivo endothelial repair capacity of early EPCs from PHAs. The eNOS was also recognized to be a source of superoxide with the BH4 deficiency. To identify the source of superoxide production in early EPCs from PHAs, the early EPCs of PHAs were treated with eNOS inhibitor l-nitro-arginine methyl ester. The data showed that l-nitro-arginine methyl ester treatment had little effect to inhibit superoxide production of early EPCs from PHAs when compared with apocynin (Figure S9). These data indicated that hyperaldosteronemia impaired in vivo endothelial repair capacity of early EPCs via increased oxidative stress derived from activating NADPH oxidase rather than uncoupling eNOS. The oxidative BH4 degradation and derangement of eNOS/NO pathway were the subsequent alteration of increased oxidative stress and took part in hyperaldosteronemia-caused functional impairment of early EPCs.

BH4 has been recognized gradually as a potential therapeutic target in atherosclerotic vascular disease for regulating eNOS activity.19 Supplementation with BH4 was able to slow the progression of atherosclerosis.28 Recently, Nagata et al21 reported that BH4 treatment prevented aldosterone-induced eNOS uncoupling of endothelial cells. In the present study, the high-performance liquid chromatography analysis showed that BH4 treatment increased intracellular BH4 content of early EPCs from PHAs, indicating that intracellular BH4 can be supplemented exogenously. We then demonstrated that the exogenous supplementation of BH4 markedly increased the eNOS dimerization and the NO generation, as well as improved in vivo endothelial repair capacity of early EPCs from PHAs, indicating that BH4 treatment has beneficial effect on modifying hyperaldosteronemia-caused functional impairment of early EPCs.

The MR that mediated the response to aldosterone expressing in early EPCs has been proved previously15,16 and was identified in the present study again (Figure S10). Spironolactone, a MR antagonist, has been generally applied in clinic for treatment with PHAs or the condition of secondary activation of renin–angiotensin–aldosterone system such as heart failure and myocardial infarction. The present study showed that oral spironolactone was able to improve in vivo endothelial repair capacity of early EPCs and endothelial function in PHAs. Oral spironolactone also decreased oxidative stress level, reduced BH4 oxidation, attenuated eNOS uncoupling, and increased NO generation in early EPCs of PHAs. These results demonstrated that hyperaldosteronemia-induced functional impairment of early EPCs was MR dependent and put forward a novel mechanism contributing to beneficial effects of MR blockade for vascular protection.

Hyperaldosteronemia whether occurred in PHAs or secondary cases such as patients with acute myocardial infarction or chronic heart failure has been proved to be adverse to cardiovascular outcome.12,29,30 The present study demonstrated the negative effect of hyperaldosteronemia on early EPC-mediated endogenous endothelial repair process via the well-established simple PHAs avoiding underlying heterogeneous interference of other cardiovascular risk, providing novel evidence to support the hyperaldosteronemia as an independent risk factor for the pathogenesis of atherosclerotic vascular disease. The present study also demonstrated that suppressing oxidative stress or exogenous supplement with BH4 was beneficial for early EPC function, suggesting that antioxidant therapy and BH4 supplementation may be novel supplementary treatment means for hyperaldosteronemia particularly in the situation where the high dose of MR antagonist should be avoided because of its side effect in long-term treatment.

Perspectives

The present study demonstrated that the hyperaldosteronemia leads to diminished early EPC-mediated endothelial repair capacity, and the effect is MR dependent. NADPH oxidase–derived oxidative stress activation and subsequently oxidative BH4 degradation, as well as eNOS uncoupling, were responsible for hyperaldosteronemia-caused early EPC dysfunction. MR antagonist, suppressing oxidative stress or exogenous BH4 supplementation, has a beneficial effect to improve early EPC-mediated endothelial repair capacity, which was impaired by hyperaldosteronemia. Discrimination and intervention of hyperaldosteronemia should draw more attention in clinic practice to prevent and retard the pathogenesis of atherosclerotic vascular disease.

Sources of Funding

This work was supported by the 973 Program (2013CB531200) and National Nature Science Foundation (31530023, 81100201, 31370941, and 81200249) of China.

Novelty and Significance

What Is New?

Endothelial repair capacity of early endothelial progenitor cells and endothelial function are substantially impaired in hypertensive patients with primary hyperaldosteronemia, and the impairment is more severe than essential hypertension patients with similar blood pressure level.

What Is Relevant?

The findings in this study demonstrate a novel mechanism for hyperaldosteronemia contributing to the development of endothelial injury and provide a new evidence to support the hyperaldosteronemia as an independent risk factor for the pathogenesis of atherosclerotic vascular disease.

Summary

The present study demonstrates for the first time that hyperaldosteronemia impairs early endothelial progenitor cell–mediated endothelial repair capacity in addition to elevating blood pressure and provides a novel therapeutic means for increased endothelial repair capacity in hypertensive patients with primary hyperaldosteronemia.